THE HELA 200KDA U5 SNRNP-SPECIFIC PROTEIN AND ITS HOMOLOG IN SACCHAROMYCES-CEREVISIAE ARE MEMBERS OF THE DEXH-BOX PROTEIN FAMILY OF PUTATIVE RNA HELICASES

The primary structure of the 200 kDa protein of purified HeLa U5 snRNP s (U5-200kD) was characterized by cloning and sequencing of its cDNA. In order to confirm that U5-200kD is distinct from U5-220kD we demonst rate by protein sequencing that the human US-specific 220 kDa protein is homologous to the yeast US-specific protein Prp8p. A 246 kDa protei n (Snu246p) homologous to U5-200kD was identified in Saccharomyces cer evisiae. Both proteins contain two conserved domains characteristic of the DEXH-box protein family of putative RNA helicases and RNA-stimula ted ATPases. Antibodies raised against fusion proteins produced from f ragments of the cloned mammalian cDNA interact specifically with the H eLa U5-200kD protein on Western blots and co-immunoprecipitate U5 snRN A and to a lesser extent U4 and U6 snRNAs from HeLa snRNPs. Similarly, U4, U5 and U6 snRNAs can be co-immunoprecipitated from yeast splicing extracts containing an HA-tagged derivative of Snu246p with HA-tag sp ecific antibodies. U5-200kD and Snu246p are thus the first putative RN A helicases shown to be intrinsic components of snRNPs. Disruption of the SNU246 gene in yeast is lethal and leads to a splicing defect in v ivo, indicating that the protein is essential for splicing. Anti-U5-20 0kD antibodies specifically block the second step of mammalian splicin g in vitro, demonstrating for the first time that a DEXH-box protein i s involved in mammalian splicing. We propose that U5-200kD and Snu246p promote one or more conformational changes in the dynamic network of RNA-RNA interactions in the spliceosome.